The first of a two part series of articles, this article discusses the development of flavor biotechnology as an alternative to organic synthesis of flavors with examples of fragrance/flavor ingredients made by using micro-organisms and fungus. The article also details trends in biotech method of producing vanillin

The smell of food during cooking is due to several biochemical processes that occur. For example, the flavor of delicious food is because of how the fats and sugars in the food interact with amino acids. Similarly when a new flavor is made, it is actually the result of the interplay of organic chemistry and biochemistry. And best of all some of these flavors are based on vegetarian ingredients.

Flavoring compounds find use in the following sectors:
• Food industry
• Perfumery industry
• Pharmaceutical industry

Generally flavoring ingredients are sourced from:
• Plants
• Or obtained through chemical synthesis

Some perfumes and flavors that tastes and smell good cannot be profitably made by organic synthesis and the aim is to make them by biosynthesis----and biotechnology helps us to understand these processes at the gene, protein or metabolite levels.

So in recent times biotechnology also has a role to play in development of flavoring compounds. Such compounds are reckoned as natural flavorings. Initially enzymes played a role in development of flavoring compounds, but now biotransformation of flavor compounds can be achieved with microbial whole cells in suitable bioreactors. However, what is important is that, flavor compounds made through biotech methods have been classified as natural. Undoubtedly the term natural has a favorable impact on the consumer.

Fermentation process is used for making flavor and perfumery ingredients using microorganisms or special enzymes. These products are dubbed natural in origin even having special properties such as optical activity. Natural ingredients are vital for food and cosmetic products especially when natural volatile flavors or what is called natural aromas are used.

There are different ways to make these flavor ingredients using biotechnology:
• Using biocatalyzed reactions
• Or through biogenetic route



Problems in making flavors through chemical synthesis and extraction from plants/animals

The flavors produced through chemical synthesis are not eco-friendly and often the compounds are derived as racemic mixtures or what is called chiral molecules. Similarly the flavor concentration in animals and plants is in the lower side and their extraction is an expensive business---both in terms of isolation and purification. Hence the importance of making flavors through biotech means.


Example of fragrance/flavor ingredients made by using micro-organisms

β-pinene and α-pinene: As precursors to several flavors and fragrances they are important. Chemically they are bicycle monoterpens hydrocarbons and constitute the largest secondary metabolites of plants.Ferula gummosa Boiss plant native to Iran is a source of monoterpene β-pinene.

These monoterpene precursors can be converted to flavors/fragrances using biotech methods as for example using microorganisms that can convert β-pinene to α-pinene. For example, β-pinene substrate can be converted to α-pinene using the microbial strain Micrococcus sp. strain PIN which is non spore forming and gram positive. This bacterial strain can be found in the Ferula galbanum plant

The bioconversion of β-pinene to α-pinene using a bacterial strain was unique but was dependent on the quantum of nitrogen levels in the medium. The optimal growth of this bacteria occurred when the following condition was maintained: nitrogen source ~7 g L-1 urea, temperature~ 30°, aeration rate ~120 rpm, pH 6.5. Making α-pinene using whole cells, enzymes and biocatalysts is a viable proposition

Similarly research is underway to convert components of other essential oils to flavor and fragrance ingredients


Example of fragrance/flavor ingredients made by using fungus

The pathogenic fungus Botrytis cinerea has been found to be successful in transforming α-pinene to predominantly 4_-hydroxy-(D)-α-pinene-6-one and some other metabolites. Similarly the honey fungus Armillariella mellea could transform (Ð)-α-pinene and (Ð)-_-pinene.


The use of yeast for developing flavor

Supposing a sugar solution that is totally devoid of any flavor is fermented with the yeast S. cerevisiae the resulting solution will have adequate flavor. Therefore yeast is used in the making of wine and beer, and biotechnology constantly strives to genetically improve these starter strains.

Even if flavors and perfumes are bio-sourced either from botanicals or through fermentation, they still need to be purified for obtaining good quality. The acceptable standards of purity and quality of flavor chemicals is specified in the Food Chemicals Codex wherein the acceptable physical attributes of the ingredients, compliance of purity requirements, and suggested storage and packaging attributes can be found.



Biotech method of producing vanillin

Vanillin which is chemically 4-hydroxy-3-methoxybenzaldehyde is a component of the aromatic flavor vanilla. This flavor is used in the food and cosmetic industries and is greatly in demand. Most of this demand is met by chemically synthesizing this compound. Natural vanillin is purely extracted from Vanilla planifolia beans. But this involves an expensive process and can only meet less than 5% of the world demand. Therefore the importance of exploring biotech means to produce vanillin especially using ferulic acid as the starting point with recombinant E.coli cells as biocatalysts. Vanillin can also be made by bioconversion of lignin, isoeugenol and other aromatic acids or through biosynthesis by using fungi and plant cells.

Example

The use of E.Coli JM109 cells expressing genes from Pseudomonas fluorescens BF13 (ferulic-acid degrader) in optimal culture conditions alongside a suitable promoter was able to sustain the conversion of ferulic acid to vanillin without producing the unwanted oxidation or reduction products downstream. Furthermore, it has been proved that by using resting cells of JM109 E.Coli strain the yields obtained in making vanillin were substantial.

Problems in making vanillin through biotech methods and how they are addressed

The micro-organisms Amycolatopsis sp. HR167 and Streptomyces setonii ATCC 39116 (actinomycetes) are capable of giving the highest yields of vanillin from ferulic acids. But processing costs are on the higher side because the growth of micro-organisms gives a viscous broth and there is the tendency to form pellets. When micro-organisms of Pseudomonas variety are used, these problems do not occur, but yield tends to be on lower side.

Recombinant strains

Vanillin has a high propensity for chemical reactions. Therefore, vanillin when produced may get transformed to vanillic acid or vanillyl alcohol. But now the aim is to prevent further oxidation of vanillin by using recombinant strains that carry the genes which encode for the transformation of ferulic acid to vanillin. This means studying the enzymes that are responsible for converting ferulic acid to vanillin and also understanding the genes that encode for this process.

Some recombinant bacterial strains have been found effective in making vanillin. For example, E. coli XL1-Blue (pSKvaomPcalAmcalB) was effective in making ferulic acid from eugenol and thereafter E. coli (pSKechE/Hfcs) was effective in converting this ferulic acid to vanillin. It has also been noted that E. coli strain which carries the Amycolatopsis genes could produce the maximum vanillin. Recombinant strains have been found to be better than natural strains in making vanillin.


Conclusion

Referring to technical literature it's clear that as of now work pertaining to identifying the biosynthetic pathways for the most important flavor volatiles has already been done. Similarly work relating to identifying the genes encoding the synthetic enzymes has also been done. Ultimately every flavor or fragrance that triggers the sense of smell and taste is the direct outcome of chemistry researching nature to create an exquisite biological response.

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PART 2: https://www.biotecharticles.com/Applications-Article/Flavor-Biotechnology-Part-2-495.html